Tryptophan enzyme mutant and application thereof

Abstract

The invention discloses a tryptophan enzyme mutant and application thereof. The tryptophan enzyme mutant is obtained by mutation of at least one site of the 50th amino acid L, the 394th amino acid V and the 395th amino acid E of a tryptophan enzyme parent. The tryptophan enzyme mutant disclosed by the invention has good substrate bias, particularly has better selectivity and catalytic efficiency on 6 '-halogenated tryptophan, and not only simplifies the production process, but also improves the production efficiency when being used for biosynthesis production of the tylosin.

Description

Technical Field

[0001] The present application relates to the technical field of tryptophanase mutation, and in particular to a tryptophanase mutant and its application. Background Art

[0002] Tyrian purple is a dye extracted from the hypobranchial glands of the Phoenician shell (Bolinus brandaris), also known as the "dye snail." This ancient and precious purple dye dates back to the ancient city of Pompeii in the Aegean Sea in 1800 BC. It earned the name "Purpura Tyri" (Latin: "Tyrian purple"), after the Phoenician city-state of Tyres, where it was primarily produced. Over time, "Tyrian purple" evolved into a more generic name. In the ancient Greek epics of Homer, a clear distinction is made between marine Tyrian purple and textiles made with other dyes. In 301 AD, Diocletian's "Price Decree" stipulated that a pound of silk dyed with Tyrian purple could be sold for as much as 150,000 Roman dinars, equivalent to approximately $3 million today. Due to its scarcity and high cost, Tyrian purple was only affordable for the wealthy and powerful, who used it to create luxurious clothing, ornaments, and royal insignia, making it a symbol of nobility and royalty.

[0003] Tyrian purple is a mixture of various compounds. The primary purplish substance was later identified as a bromine-containing organic compound: 6,6'-dibromoindigo (6BrIG), with other minor components including 6-bromoindigo, indigo, 6,6'-dibromoindigo, and monobromoindigo. The production process for Tyrian purple is extremely complex and stinks. It begins with the collection of specialized glandular shells from the Mediterranean region, followed by a series of treatments, including oxidation and reduction reactions, to obtain pure Tyrian purple dye. This process is time-consuming, labor-intensive, and yields extremely limited results. It is estimated that 12,000 shells are needed to produce 1.4 grams of dye, barely enough to dye the edge of a single garment. To dye a Roman toga, approximately 250,000 shells would be needed to produce half an ounce of dye. Chemical synthesis is extremely difficult to introduce two bromine atoms into the indole precursor. Current methods, which involve bromination using bromine gas or hydrogen bromide, lack site-specificity, resulting in low yields of Tyrian purple and potential environmental hazards.

[0004] Reports, such as those in EP4242311A1, CN115851847A, and CN115992185A, have reported the synthesis of Tyrian purple using a biosynthetic method. This involves a two-step reaction: halogenation of tryptophan to produce a 6'-halogenated tryptophan precursor, followed by hydrolysis of the 6'-halogenated tryptophan precursor to produce 6,6'-dibromoindigo (6BrIG), the main component of Tyrian purple. This method offers advantages such as environmental friendliness, high biosafety, and high production efficiency. EP4242311A1 and CN115992185A employ two separate reactions, followed by fermentation to form water-insoluble pigment particles. While feasible on a small scale, pilot-scale or industrial production suffers from issues such as pigment sticking to the wall and high cleaning costs, making large-scale production difficult. The CN115850847A process glycosidates 6,6'-dibromoindigo (6BrIG), ultimately forming a water-soluble halogenated glycoside. After fermentation, the halogenated glycoside in the supernatant needs only to be collected. 6BrIG can be obtained by removing the glycoside when it is used. This process is more feasible, more operational, and less costly for large-scale production of Tyrian purple pigment.

[0005] Because the Tyrian purple precursor, 6'-halogenated tryptophan, is expensive, these biosynthetic methods primarily use tryptophan and sodium bromide or sodium chloride as precursor substrates, first halogenating the tryptophan to produce the 6'-halogenated tryptophan. However, tryptophanase (TnaA) lacks specific selectivity for tryptophan and 6'-halogenated tryptophan substrates. The resulting pigment is a mixture of various components, including indigo, 6-bromoindigo, 6,6'-dibromoindigo, 6,6'-dibromoindigo, and monobromoindigo, rather than Tyrian purple, with 6,6'-dibromoindigo as its primary component. Currently, no methods have been reported for specific recognition and catalysis of 6'-halogenated tryptophan by tryptophanase. Therefore, the halogenation reaction and the 6'-halogenated tryptophan hydrolysis reaction require separate tanks. Typically, fermentation is performed first to produce the 6'-halogenated tryptophan, and then the fermentation supernatant is collected for the second step of 6'-halogenated tryptophan hydrolysis and fermentation. This results in a complex and costly process.

[0006] Therefore, how to improve the specific selectivity of tryptophanase is a key factor affecting the biosynthesis of Tyrian purple, and is also the research focus and difficulty in the field of Tyrian purple biosynthesis. Summary of the Invention

[0007] The purpose of this application is to provide a new tryptophanase mutant and its application.

[0008] This application adopts the following technical solutions:

[0009] The first aspect of the present application discloses a tryptophanase mutant obtained by mutation of at least one of the 50th amino acid L, the 394th amino acid V and the 395th amino acid E of a tryptophanase parent.

[0010] It should be noted that the tryptophanase parent in this application refers to a wild-type tryptophanase without mutations. The present application discovered that the three sites of the tryptophanase parent, L at position 50, V at position 394, and E at position 395, namely LEU50 (leucine), VAL394 (valine), and GLU395 (glutamate), can affect substrate preference; therefore, by performing different mutations on these three sites, tryptophanase mutants with different substrate preferences can be obtained. For example, one of the three sites can be mutated, or any two of them can be mutated, or all three can be mutated simultaneously.

[0011] In one implementation of the present application, the 50th amino acid L is mutated to A, V, I, N, S, G, T, C, H, F or M.

[0012] In one implementation of the present application, the amino acid V at position 394 is mutated to I, S, A, P, T, H, G, N, L or F.

[0013] In one implementation of the present application, the amino acid E at position 395 is mutated to S, G, C, A, E, T, P or D.

[0014] It should be noted that the above mutations of amino acid L at position 50, amino acid V at position 394 or amino acid E at position 395 are the specific mutation types used in the embodiments of the present application; it can be understood that under the inventive concept of the present application, it is not ruled out that other mutation types can also be used to obtain tryptophanase mutants with different substrate preferences.

[0015] In one implementation of the present application, the tryptophanase mutant is obtained by simultaneously mutating the 50th amino acid L, the 394th amino acid V, and the 395th amino acid E of the tryptophanase parent.

[0016] It should be noted that the present application found that the three sites LEU50, VAL394, and GLU395 can affect substrate bias, and that simultaneous mutation of the three sites can obtain a tryptophanase mutant with a substrate bias effect; therefore, in a preferred embodiment, the present application mutates the three sites simultaneously to obtain the preferred tryptophanase mutant of the present application.

[0017] In one implementation of the present application, the specific mutation types of the simultaneous mutations of amino acid L at position 50, amino acid V at position 394, and amino acid E at position 395 are shown in Table 1.

[0018] In one implementation of the present application, the specific mutation types of the preferred simultaneous mutations of amino acid L at position 50, amino acid V at position 394, and amino acid E at position 395 are shown in Table 2.

[0019] It should be noted that the tryptophanase mutants in Tables 1 and 2 are the tryptophanase mutants with a 6'-halogenated tryptophan preference demonstrated in the Examples of this application. In particular, the tryptophanase mutants in Table 2 exhibit enhanced substrate preference. It is understood that, except for the specifically indicated mutation sites, the tryptophanase mutants of this application share the same amino acids as the parent tryptophanase.

[0020] In one implementation of the present application, the tryptophanase parent is the sequence shown in SEQ ID NO.12.

[0021] The second aspect of the present application discloses a nucleic acid encoding the tryptophanase mutant of the present application.

[0022] The nucleic acid encoding the tryptophanase mutant of the present application can refer to the existing nucleic acid sequence encoding the tryptophanase parent. On this basis, the nucleic acid sequence of the tryptophanase mutant can be determined according to the specific mutation type.

[0023] The third aspect of the present application discloses a recombinant plasmid containing the nucleic acid of the present application.

[0024] It should be noted that the recombinant plasmid containing the nucleic acid of the present application can be transformed into an engineered bacterium to express the tryptophanase mutant of the present application, thereby being used to prepare Tyler purple pigment.

[0025] The fourth aspect of the present application discloses a microorganism transformed with the recombinant plasmid of the present application.

[0026] It should be noted that the microorganisms transformed with the recombinant plasmid of the present application are actually engineered bacteria used to express the tryptophanase mutant of the present application, including but not limited to conventional Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, etc.

[0027] In one implementation of the present application, the tryptophanase mutant is fused with the flavin monooxygenase MaFMO via a linker and co-expressed.

[0028] It should be noted that tryptophanase mutants can be co-expressed with flavin monooxygenases (MaFMO) from different species via a linker. The resulting engineered bacteria can ferment and synthesize 6,6'-dibromoindigo in a single step. For example, the amino acid sequence of the Tyrian purple powder strain, tnaA+-FL-MaFMO, is fused and expressed, as shown in SEQ ID NO. 18. This sequence only illustrates one tryptophanase mutant; the remaining tryptophanase mutants can be modified in sequence based on the specific mutation type.

[0029] In one implementation of the present application, the tryptophanase mutant is fused and co-expressed with the flavin monooxygenase MaFMO-UGT via a linker.

[0030] It should be noted that the engineered strain obtained by co-expressing a tryptophanase mutant through a linker fusion with MaFMO-UGT can ferment and synthesize the glycoside form of 6,6'-dibromoindigo in a single step. The glycoside form is highly soluble in water and can be secreted extracellularly. For example, the sequence of the strain expressing the pyruvate glycoside is: Ptac-tnaA+-FL-fmo-Ptac-UGT, as shown in SEQ ID NO. 19. The sequence shown in SEQ ID NO. 19 only illustrates one tryptophanase mutant. Other tryptophanase mutants can be modified according to the specific mutation type.

[0031] The fifth aspect of the present application discloses the use of the tryptophanase mutant, the nucleic acid, the recombinant plasmid or the microorganism of the present application in the preparation of Tyrian purple pigment.

[0032] It should be noted that the microorganisms of this application are engineered bacteria capable of expressing the tryptophanase mutants of this application and, therefore, can be used to prepare Tyler purple pigment. Of course, depending on the design, the tryptophanase mutants of this application can be fused and co-expressed with MaFMO or MaFMO-UGT. The nucleic acids and recombinant plasmids of this application can be used as raw materials to prepare engineered bacteria expressing the tryptophanase mutants of this application. Once the tryptophanase mutants of this application have been obtained, they can, of course, be used directly to prepare Tyler purple pigment.

[0033] The sixth aspect of the present application discloses Tyler purple pigment prepared using the tryptophanase mutant, nucleic acid, recombinant plasmid or microorganism of the present application.

[0034] It should be noted that the Tyler purple pigment prepared by the tryptophanase mutant, the nucleic acid, the recombinant plasmid or the microorganism of the present application has a higher purity and quality due to the good 6'-halogenated tryptophan preference of the tryptophanase mutant of the present application, that is, the product has 6,6'-dibromoindigo as the main component.

[0035] The seventh aspect of the present application discloses a method for screening a 6'-halogenated tryptophan-biased tryptophanase mutant, comprising the following steps:

[0036] In the protein model construction step, the molecular 3D structures of tryptophanase, coenzyme pyridoxal phosphate and tryptophan were docked using software to obtain the protein model of TnaA+PLP+Trp;

[0037] The mutation site analysis step includes using software to modify the TnaA+PLP+Trp model and analyze the mutation site;

[0038] The mutation library construction step involves performing saturation random mutagenesis on the mutation sites obtained through analysis to obtain a tryptophanase mutation library;

[0039] In the tryptophanase mutant screening step, the tryptophanase mutant library is screened at least once using a tryptophan substrate to obtain a tryptophanase mutant with a 6'-halogenated tryptophan preference.

[0040] It should be noted that saturated random mutagenesis in this application refers to the random combination of the sites obtained by screening according to different mutation types, for example, the three sites of LEU50, VAL394, and GLU395, designing one of the three sites to undergo any mutation, any two of the three sites to undergo any mutation in any combination, and any three sites to undergo any mutation in any combination at the same time, thereby forming a tryptophanase mutation library covering all possible mutations.

[0041] The beneficial effects of this application are:

[0042] The tryptophanase mutant of the present application has good substrate preference, especially better selectivity and catalytic efficiency for 6'-halogenated tryptophan. When used in the biosynthesis of Tyrian purple pigment, it not only simplifies the production process but also improves the production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a diagram showing the result of tryptophanase molecular docking in the examples of this application;

[0044] Figure 2 and Figure 3 This is a diagram showing the comparison results of tnaA chloro-substitution screening sequencing in the examples of this application;

[0045] Figure 4 and Figure 5 This is a diagram showing the alignment results of tnaA bromosubstitution screening sequencing in the examples of this application;

[0046] Figure 6 This is a photo of some shake flask fermentation rescreening results of the substrate preference test in the examples of this application;

[0047] Figure 7 This is a photo of some shake flask fermentation rescreening results of 6'-halotryptophan-preferring strains in the examples of this application;

[0048] Figure 8 is the standard curve of L-tryptophan in the examples of this application;

[0049] Figure 9 is the standard curve of 6-chloro-L-tryptophan in the examples of this application;

[0050] Figure 10is the standard curve of 6-bromo-L-tryptophan in the examples of this application;

[0051] Figure 11 The test results of the residual L-tryptophan and tryptophan chloride content in the examples of the present application;

[0052] Figure 12 The test results of the residual L-tryptophan and brominated tryptophan substrates after the reaction in the examples of the present application are shown in FIG.

[0053] Figure 13 This is a fermentation photo of the one-step synthesis of Tyrian purple powder with a preference for 6'-halogenated tryptophan in the embodiment of the present application by the engineering bacteria 040+ in a 10L tank;

[0054] Figure 14 This is a fermentation photo of the 10L tank fermentation of the engineering bacteria 040+-UGT for the one-step synthesis of 6'-halogenated tryptophan-biased tyrpuroside in the embodiment of the present application;

[0055] Figure 15 This is the dichloroindigo standard curve obtained by detecting the standard stock solution with an enzyme marker in the examples of the present application. DETAILED DESCRIPTION

[0056] Since there are currently no studies or reports on tryptophanase specifically recognizing and catalyzing 6'-halogenated tryptophan, the present application creatively conducts a semi-rational design of tryptophanase by using a directed evolution approach to perform saturation random mutagenesis on amino acid sites related to 6'-halogenated tryptophan to obtain tryptophanase mutants with a significant preference for 6'-halogenated tryptophan.

[0057] This application utilizes Autoduck-vina 1.1.2, pymol software, and Autodock Tools 1.5.7 software to model the tryptophanase TnaA as a receptor and different tryptophan substrates as ligands, and to determine the sites in the model that influence substrate preference. Analysis determined that three sites, LEU50 (leucine), VAL394 (valine), and GLU395 (glutamate), can influence substrate preference. Saturation random mutagenesis was performed on these three sites, and 60 three-point mutants with substrate selectivity were screened. Fourteen tryptophanases with a 6'-halogenated tryptophan substrate preference were preferably obtained. In addition, the present application co-transforms the obtained mutant plasmid with the tryptophan-6 halogenase plasmid or the tryptophan-6 halogenase-glycosidase plasmid into Escherichia coli. Only one strain is needed for fermentation, and tryptophan and sodium bromide precursors are added. The biosynthesis of the toner or glycoside form of 6,6'-dibromoindigo can be completed in a one-step fermentation process, which greatly simplifies the process, reduces the fermentation cost, and has significant industrial potential.

[0058] The semi-rational design of the present application conducts substrate-biased directed evolution of tryptophanase, comprising the following steps:

[0059] Through literature research and homologous protein comparisons, molecular docking using AutoDock-vina and pymol software generated a protein model of TnaA+PLP+Trp. Analysis showed that the Trp 6 position is located near LEU50 (leucine), PHE39 (phenylalanine), VAL394 (valine), and GLU395 (glutamic acid). Because PHE39 (phenylalanine) is a structurally stable benzene ring, this application only considered random mutations of the other three amino acids, namely LEU50 (leucine), VAL394 (valine), and GLU395 (glutamic acid). Through three-site saturation random mutagenesis, a tnaA mutant library was constructed. Then, different tryptophan substrates were added for primary and secondary screening experiments to verify the results, ultimately obtaining mutants with a 6'-halogenated tryptophan preference.

[0060] This application has developed for the first time a directed evolution strategy for the substrate preference of tryptophanase, and applied this strategy to modify the substrate preference of tryptophanase. Combining three-site saturation mutation and combinatorial mutation technology, an enzyme mutant with a 6'-halogenated tryptophan substrate preference was successfully obtained. Furthermore, this application also co-expressed the mutant with modified preference by fusing it with flavin monooxygenase MaFMO from different species through Linker. The obtained single bacteria can ferment and synthesize the color powder form of 6,6'-dibromoindigo in one step. By co-expressing the fusion of Linker and MaFMO-UGT, the obtained single bacteria can ferment and synthesize the glycoside form of 6,6'-dibromoindigo in one step. The glycoside form is easily soluble in water and can be secreted outside the cell. The fermentation product is simple to collect, which greatly simplifies the process, reduces the fermentation cost, and has significant industrialization potential. It has broad development prospects in the industrial application of biocatalytic production of Tyler purple pigment.

[0061] The present invention is further described in detail below by means of specific examples. The following examples are only provided to further illustrate the present invention and should not be construed as limiting the present invention. Unless otherwise specified, the methods for constructing plasmids or strains in this application are based on the Molecular Cloning Laboratory Manual (4th edition).

[0062] Example 1

[0063] The mutation site of tryptophanase TnaA was determined. The specific steps are as follows:

[0064] 1. Download and process the 3D structures of PLP (pyridoxal coenzyme phosphate) and Trp (tryptophan): Download the 3D structures of the molecules from the TCMSP website. Use pymol and AutodockTools 1.5.7 software to perform full hydrogen addition and charge calculation operations, and save them in PDBQT format for future use.

[0065] 2. Download and processing of the 3D structure of TnaA (tryptophanase): The 3D structure of TnaA was downloaded from the PDB website (PDB DOI: https: / / doi.org / 10.2210 / pdb2C44 / pdb). Pymol and AutodockTools 1.5.7 were used to remove water molecules and metal ions, add polar hydrogens, and calculate charges. The structure was then saved in PDBQT format for future use.

[0066] 3. Molecular Docking: Molecular docking was performed using Autoduck-vina 1.1.2 software, using the PDBQT format file of TnaA as the receptor and the PDBQT format file of PLP as the ligand. The resulting docking was named TnaA-PLP. Water molecules and metal ions were removed, polar hydrogens were added, and charges were calculated for TnaA-PLP using pymol and Autodock Tools 1.5.7 software. The resulting docking was saved in PDBQT format for future use. Molecular docking was performed using Autoduck-vina 1.1.2 software, using the PDBQT format file of TnaA-PLP as the receptor and the PDBQT format file of Trp as the ligand. The resulting docking was named TnaA-PLP-Trp.

[0067] 4. Manual docking: Based on the literature Structure of Escherichia coli tryptophanase (doi: 10.1107 / S0907444906019895), the TnaA-PLP-Trp model was modified using pymol software. After analysis, the three sites LEU50 (leucine), VAL394 (valine), and GLU395 (glutamic acid) were identified as the sites for modification. The results are as follows Figure 1 shown. Figure 1 This is the result of tryptophanase molecular docking.

[0068] Example 2

[0069] The synthesis of the tnaA gene mutation library and the construction of the mutant plasmid library strain were carried out as follows:

[0070] 1. The TnaA amino acid sequence, i.e., the tryptophanase parent sequence shown in SEQ ID NO. 12, was sent to Suzhou Silicon-Based Biotechnology Co., Ltd. for random mutation of the bases at three sites of the TnaA protein, LEU50 (leucine), VAL394 (valine), and GLU395 (glutamic acid), to synthesize the tnaA gene mutation library.

[0071] SEQ ID NO.12:

[0072] MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDSEDVFID L LTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYALAESVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKREQEKGLDRSKMVAFSNYFFDT TQGHSQINGCTVRNVYIKEAFDTGVRYDFKGNFDLEGLERGIEEVGPNNVPYIVATITSNSAGGQPVSLANLKAMYSIAKKYDIPVVMDSARFAENAYFIKQREAEYKDWTIEQITRETYKYADMLA MSAKKDAMVPMGGLLCMKDDSFFDVYTECRTLCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLDWLAYRIAQVQYLVDGLEEEIGVVCQQAGGHAAFVDAGKLLPHIPADQFPAQALACELYKVAGIRA VE IGSFLLGRDPKTGKQLPCPAELLRLTIPRATYTQTHMDFIIEAFKHVKENAANIKGLTFTYEPKVLRHFTAKLKEV

[0073] In the sequence shown in SEQ ID NO. 12, the amino acids marked with single underlines are the sites to be mutated: LEU50, VAL394, and GLU395.

[0074] 2. Construction of mutant plasmid library: The tnaA gene mutation library product delivered by Suzhou Silicon-Based Biotechnology Co., Ltd. was amplified by PCR using primers tnaA-BsaⅠ-F (SEQ ID NO.1) and tnaA-BsaⅠ-R (SEQ ID NO.2).

[0075] SEQ ID NO.1: 5'-aatGGTCTCGCTAGatggaaaactttaaacatctccctgaaccg-3'

[0076] SEQ ID NO.2: 5'-ACAGGTCTCGTCTCaacttctttcagttttgcggtgaagtg-3'

[0077] The PCR reaction system was 50 μL, including: 2×Lamp mastermix 25 μL, tnaA gene mutation library 1 μL, tnaA-BsaⅠ-F 1 μL, tnaA-BsaⅠ-R 1 μL, and ddH2O 22 μL.

[0078] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 2 min, annealing at 62°C for 56 s, and extension at 72°C for 43 s. After the cycle, final extension was performed at 72°C for 5 min, followed by standby at 10°C.

[0079] After PCR amplification, gel electrophoresis was used for verification as follows: 1% agarose gel was prepared; 5 μL of PCR product was taken, and 5000 bp DNA Maker was used as a reference; electrophoresis was performed at 140 V for 20 min.

[0080] The PCR product with the target band of approximately 1441 bp was recovered for future use. Using the pW1-Ptac-RiboJ-BsaI plasmid and the recovered PCR product from the tnaA gene mutation library, the pW1-Ptac-tnaA+ (SEQ ID NO. 13) plasmid library was constructed using the Golden Gate method. The Golden Gate method steps and conditions are as follows, as per the Molecular Cloning Manual (4th edition):

[0081] 20μL Golden gate reaction system includes: Bsa1-HFv21μL, T4 ligation 1μL, 10×T4 ligation buffer

[0082] 2 μL, 1 μL of 200 ng / μL PW1 plasmid, 0.2 μL of 100 ng / μL Pcr-010 fragment, and 14.8 μL ddH2O.

[0083] Golden gate reaction conditions: 30 cycles: 37°C for 2 min, 16°C for 2 min, after which 37°C for 10 min, 60°C for 5 min, and then standby at 10°C.

[0084] Conversion:

[0085] a. Add 10 μL of Golden gate reaction product to 50 μL of DH5α competent medium and incubate on ice for 30 minutes;

[0086] b. Heat shock at 42°C for 90 seconds, followed by an ice bath for 5 minutes;

[0087] c. Add 500 μL of antibiotic-free LB medium and incubate at 37°C, 220 rpm, on a shaking platform for 1 hour;

[0088] d. Centrifuge and discard the supernatant, resuspend the cells, and apply all the culture to LB plates with 50 μg / mL kanamycin. Incubate at 37°C overnight.

[0089] Strain PCR verification:

[0090] a. From the transformation plate, pick a single colony and transfer it to 10 μL of sterile water. Mix thoroughly by pipetting. Take 2 μL of the bacterial solution as a template. In this example, three single colonies were selected for PCR verification. PCR amplify the tnaA gene fragment of the marked single colony using primers tnaA-BsaⅠ-F (SEQ ID NO. 1) and tnaA-BsaⅠ-R (SEQ ID NO. 2). The PCR reaction system and conditions are as described in "2. Construction of Mutant Plasmid Library." The target band is approximately 1441 bp.

[0091] b. Add 1 mL of LB culture medium containing 50 μg / mL kanamycin to the remaining 8 μL and culture at 37°C, 220 rpm, and shake for 8 h.

[0092] c. After the PCR results in the correct target band, part of the corresponding bacterial solution was stored in glycerol and part was sent to BGI for sequencing. SEQ ID NO.13:

[0093]

[0094]

[0095] The sequence shown in SEQ ID NO. 13 is the nucleic acid sequence of pW1-Ptac-tnaA+, using the parent tryptophanase as an example. For different tryptophanase mutants, the corresponding sequence can be modified. In the sequence, the dotted underline indicates the Ptac promoter region, the double underline indicates the RiboJ-RBS, the single underline indicates tnaA (using the tnaA parent as an example), and the wavy underline indicates the terminator region.

[0096] The pW1-Ptac-tnaA+ plasmid library was constructed using the Golden Gate method and transformed into the existing competent strain DH5α(DtnaA)::pSEV331-Ptac-fmo (SEQ ID NO.14) to obtain the DH5α(DtnaA)::pW1-Ptac-tnaA+, pSEV331-Ptac-fmo double-plasmid strain for subsequent mutant screening.

[0097] The construction process of the dual-plasmid strain is as follows:

[0098] (1) Prepare competent cells of the strain DH5α(DtnaA)::pSEV331-Ptac-fmo by chemical method. The plasmid carrying this strain is chloramphenicol Cm resistance. For specific methods, refer to the Molecular Cloning Laboratory Manual (4th edition);

[0099] (2) The pW1-Ptac-tnaA+ plasmid library was constructed using the Golden Gate method, all of which conferred kanamycin resistance.

[0100] (3) During transformation, an LB plate containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol was coated, and the single colony obtained by screening was the DH5α(DtnaA)::pW1-Ptac-tnaA+, pSEV331-Ptac-fmo double-plasmid strain.

[0101] SEQ ID NO.14:

[0102]

[0103]

[0104] In the sequence shown in SEQ ID NO. 14, the underlined dotted line indicates the Ptac promoter region, the double underline indicates the RiboJ-RBS, the single underline indicates the fmo, and the wavy underline indicates the terminator region.

[0105] Example 3

[0106] Tryptophanase TnaA mutant library, plate initial screening, specific steps are as follows:

[0107] 1.pW1-Ptac-tnaA+ plasmid library Cl substitution plate screening

[0108] (1) The DH5α(DtnaA)::pW1-Ptac-tnaA+, pSEV331-Ptac-fmo double-plasmid bacteria obtained in Example 2 were spread on a 30 mL LB solid plate containing 0.1 mM IPTG+0.5 mM 6Cl-Trp+0.5 mM Trp+50 μg / mL chloramphenicol+50 μg / mL kanamycin, and cultured in a 37°C incubator overnight. The plate was taken out and placed in a room temperature environment, and the color was observed after 24 hours.

[0109] (2) Select a monoclonal strain with obvious purple color on a clean bench, dilute and resuspend it with 10 μL of sterile water to obtain a resuspended bacterial solution.

[0110] (3) Take 2 μL of the resuspended bacterial solution and spot it on a 30 mL LB solid plate containing 0.1 mM IPTG + 0.5 mM 6Cl-Trp + 0.5 mM Trp + 50 μg / mL chloramphenicol + 50 μg / mL kanamycin, named plate 1.

[0111] (4) Take 2 μL of the resuspended bacterial solution and spot it on a 30 mL LB solid plate containing 0.1 mM IPTG + 1 mM 6Cl-Trp + 50 μg / mL chloramphenicol + 50 μg / mL kanamycin, named plate 2.

[0112] (5) Take 2 μL of the resuspended bacterial solution and spot it on a 30 mL LB solid plate containing 0.1 mM IPTG + 1 mM Trp + 50 μg / mL chloramphenicol + 50 μg / mL kanamycin, named plate 3.

[0113] (6) Culture in a 37°C incubator overnight, then take out and place at room temperature for 24 hours before observing the color.

[0114] (7) Mark the monoclonal strains that are purple on plate 1, purple or purple-red on plate 2, and nearly colorless on plate 3. Use primers tnaA-BsaⅠ-F and tnaA-BsaⅠ-R to PCR amplify the tnaA gene fragment of the marked monoclonal strains, and send the PCR products to a biological company for sequencing.

[0115] 2.pW1-Ptac-tnaA+ plasmid library Br replacement plate screening

[0116] (1) The DH5α(DtnaA)::pW1-Ptac-tnaA+, pSEV331-Ptac-fmo double-plasmid bacteria obtained in Example 2 were spread on a 30 mL LB solid plate containing 0.1 mM IPTG+0.5 mM 6Br-Trp+0.5 mM Trp+50 μg / mL chloramphenicol+50 μg / mL kanamycin, cultured in a 37°C incubator overnight, then taken out and placed in a room temperature environment, and the color was observed after 24 h.

[0117] (2) Select a monoclonal strain with obvious purple color on a clean bench, dilute and resuspend it with 10 μL of sterile water to obtain a resuspended bacterial solution.

[0118] (3) Take 2 μL of the resuspended bacterial solution and spot it on a 30 mL LB solid plate containing 0.1 mM IPTG + 0.5 mM 6Br-Trp + 0.5 mM Trp + 50 μg / mL chloramphenicol + 50 μg / mL kanamycin, named plate 4.

[0119] (4) Take 2 μL of the resuspended bacterial solution and spot it on a 30 mL LB solid plate containing 0.1 mM IPTG + 1 mM 6Br-Trp + 50 μg / mL chloramphenicol + 50 μg / mL kanamycin, named plate 5.

[0120] (5) Take 2 μL of the resuspended bacterial solution and spot it on a 30 mL LB solid plate containing 0.1 mM IPTG + 1 mM Trp + 50 μg / mL chloramphenicol + 50 μg / mL kanamycin, named plate 6.

[0121] (6) Culture in a 37°C incubator overnight, then take out and place at room temperature for 24 hours before observing the color.

[0122] (7) Mark the monoclonal strains that are purple on plate 4, purple or purple-red on plate 5, and nearly colorless on plate 6. Use primers tnaA-BsaⅠ-F and tnaA-BsaⅠ-R to PCR amplify the tnaA gene fragment of the marked monoclonal strains, and send the PCR products to a biological company for sequencing.

[0123] The statistical results of partial plate screening, chlorine substitution and bromine substitution plate screening are shown in Table 1.

[0124] Table 1 Statistical results of some plate screening

[0125] Screening for halogens Number of tablets Number of colonies / plate Number of colony sequencing Number of mutants Effective mutants Chlorine substitution screening 6 124 70 51 10 Bromine substitution screening 3 124 10 9 4

[0126] Colonies that may have a certain degree of preference for halogenated tryptophan were picked from the screened plates and sequenced and compared, and finally effective mutants were screened. The number of effective mutants obtained by screening is shown in Table 1.

[0127] Sequencing colony selection criteria: purple monoclonal colonies on plate 1 or plate 4, purple or purple-red on plate 2 or plate 5, and nearly colorless monoclonal colonies on plate 3 or plate 6.

[0128] Sequencing primers: primers tnaA-BsaⅠ-F and tnaA-BsaⅠ-R

[0129] Sequencing results comparison:

[0130] (1) First compare the nucleic acid sequencing results, and then convert the nucleic acid results of the mutation site into amino acid sequences;

[0131] (2) Compare the amino acid mutation sequence results to obtain the mutant amino acid sequence.

[0132] Some sequencing results are as follows Figures 2 to 5 As shown, Figure 2 and Figure 3 This is the colony selected and sequenced for tnaA chloro-substitution, with the alignment results of amino acids at positions 50, 394, and 395, marked in yellow. Figure 4 and Figure 5 This is a colony screened for tnaA bromosubstitution, with alignment results for amino acids at positions 50, 394, and 395, highlighted in yellow. Excluding mutants with duplicate sequencing, 51 tryptophanase mutants were identified by chlorine substitution and 9 by bromine substitution, for a total of 60 mutants, as shown in Table 2.

[0133] Table 2 Specific mutation types of 60 tryptophanase mutants

[0134]

[0135]

[0136] On plates 1 or 4, only halogenated tryptophan is used as a substrate; on plates 2 or 5, both halogenated tryptophan and tryptophan are present; and on plates 3 or 6, only tryptophan is used as a substrate. If the colony turns purple on the plate containing halogenated tryptophan but does not change color on the plate containing only tryptophan, this indicates that the mutant's tryptophanase preferentially utilizes the halogenated tryptophan substrate and is unable to utilize unhalogenated tryptophan. The tryptophanase mutant corresponding to this colony is considered a valid mutant. This screening ultimately yielded 10 valid tryptophanase mutants derived from chlorine substitutions and 4 valid mutants derived from bromine substitutions, for a total of 14 valid mutants, as shown in Table 3.

[0137] Table 3 14 effective mutants

[0138]

[0139] In Tables 2 and 3, the "Mutant" column corresponds to the tryptophanase mutant number, the "LEU50" column corresponds to the mutation type of amino acid L at position 50, the "VAL394" column corresponds to the mutation type of amino acid V at position 394, and the "GLU395" column corresponds to the mutation type of amino acid E at position 395. For example, Mutant 1 refers to a tryptophanase mutant obtained by mutating amino acid L at position 50 to N, amino acid V at position 394 to A, and amino acid E at position 395 to A. This refers to a tryptophanase mutant obtained by mutating amino acid L at position 50 to N, amino acid V at position 394 to A, and amino acid E at position 395 to A based on the tryptophanase parent sequence shown in SEQ ID NO. 12, as shown in SEQ ID NO. 20. Therefore, Mutant 1 can also be represented as an NAA mutant, i.e., the mutant is represented by three amino acid mutation types, and the rest are similar.

[0140] SEQ ID NO.20:

[0141] MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDSEDVFID N LTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYALAESVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKREQEKGLDRSKMVAFSNYFFDTTQGHSQINGCTVRNVYIKEAFDTGVRYDFKGNFDLEGLERGIEEVGPNNVPYIVATITSNSAGGQPVSLANLKAMYSIAKKYD IPVVMDSARFAENAYFIKQREAEYKDWTIEQITRETYKYADMLAMSAKKDAMVPMGGLLCMKDDSFFDVYTECRTLCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLDWLAYRIAQVQYLVDGLEEIGVVCQQAGGHAAFVDAGKLLPHIPADQFPAQALACELYKVAGIRA AA IGSFLLGRDPKTGKQLPCPAELLRLTIPRATYTQTHMDFIIEAFKHVKENAANIKGLTFTYEPKVLRHFTAKLKEV

[0142] It is understood that the sequences of the remaining tryptophanase mutants can be adjusted to the corresponding mutation types by referring to Table 2, and will not be described here in detail.

[0143] Example 4

[0144] Construction of a color-engineered strain with a preference for 6'-halogenated tryptophan and verification by shake flask screening. The specific steps are as follows:

[0145] 1. Construction of a color powder engineering bacterial strain for the one-step fermentation synthesis of 6,6'-dibromoindigo

[0146] (1) PCR amplification of the strain pW1-Ptac-tnaA+ obtained from the initial plate screening was performed using primers Linker-tanA-F (SEQ ID NO. 3) and Linker-tanA-R (SEQ ID NO. 4). The products were recovered by 1% agarose gel electrophoresis and used as standby. The gel-recovered products were named Linker-tnaA+. The gel-extraction recovery kit used in this example was the TIANgel Midipurification Kit DP209. The PCR amplification reaction system and reaction conditions for Linker-tanA-F and Linker-tanA-R were the same as those for tnaA-BsaⅠ-F and tnaA-BsaⅠ-R.

[0147] SEQ ID NO.3:

[0148] 5'-TCTAGAGAAAGAGGAGAAATACTAGatggaaaactttaaacatctccctgaac-3'

[0149] SEQ ID NO.4: 5'-caCCTcctcctgagcctccaccgccaacttctttcagttttgcggtgaag-3'

[0150] (2) PCR amplification of the pSEV331-Ptac-fmo plasmid was performed using primers Linker-fmo-F (SEQ ID NO. 5) and Linker-fmo-R (SEQ ID NO. 6). The product was recovered by 1% agarose gel electrophoresis and then used for future use (the gel-recovered product was named Linker-fmo). The gel-extraction and recovery kit used in this example was the TIANgel Midi Purification Kit DP209. The PCR amplification reaction system and reaction conditions for Linker-fmo-F and Linker-fmo-R were the same as those for tnaA-BsaI-F and tnaA-BsaI-R.

[0151] SEQ ID NO.5:

[0152] 5'-ggcggtggaggctcaggaggAGGtggctcaatggccactaggattgctatcttag-3'

[0153] SEQ ID NO.6:

[0154] 5'-CTAGTATTTCTCCTCTTTCTCTAGATTAAACAAAATTATTTG-3'

[0155] (3) Linker-tnaA+ and Linker-fmo were assembled using the Gibson assembly method to obtain a ligation plasmid named pSEV331-Ptac-tnaA+-FL-fmo (SEQ ID NO. 15). For details, please refer to the Molecular Cloning Laboratory Manual (4th edition).

[0156] SEQ ID NO.15:

[0157]

[0158]

[0159] The sequence shown in SEQ ID NO. 15 is the nucleic acid sequence of pSEV331-Ptac-tnaA+-FL-fmo, using one tryptophanase mutant as an example. For different tryptophanase mutants, the corresponding sequence can be modified. In the sequence, the underlined dashed line indicates the Ptac promoter region, the double underlined line indicates the RiboJ-RBS, the single underline indicates the tnaA mutant, the bold underlined dashed line indicates the linker, the double wavy underline indicates the fmo, and the single wavy underline indicates the terminator region.

[0160] (4) The strain DH5α(DtnaA)::fre-FL-sttH (SEQ ID NO. 16) was prepared into a competent bacterium, according to the Molecular Cloning Laboratory Manual (4th edition). This strain can perform a halogenation reaction on the 6th position of tryptophan. The strain DH5α(DtnaA)::fre-FL-sttH (SEQ ID NO. 16) is referenced in patent application CN115851847A.

[0161] SEQ ID NO.16:

[0162]

[0163]

[0164] In the sequence shown in SEQ ID NO. 16, the underlined dotted line indicates the Ptac promoter region, the double underline indicates RiboJ-RBS, the single underline indicates fre, the bold underlined dotted line indicates Linker, the double underline indicates sttH, and the single wavy underline indicates the terminator region.

[0165] (5) The assembled plasmid pSEV331-Ptac-tnaA+-FL-fmo was then transformed into DH5α(DtnaA)::fre-FL-sttH. The detailed transformation steps and parameters were referred to the Molecular Cloning Laboratory Manual (4th Edition). The resulting double-plasmid recombinant strain was named 040+. This series of strains can use tryptophan and sodium chloride or sodium bromide as precursors to ferment and synthesize Tyrian purple powder products in one step. The double-plasmid recombinant strain in this example is DH5α(DtnaA)::fre-FL-sttH, pSEV331-Ptac-tnaA+-FL-fmo.

[0166] 2. Substrate preference shake flask fermentation rescreening

[0167] (1) Take 10 μL of the 040+ strain stored at -80°C and inoculate it into 4 mL of liquid LB medium (containing a final concentration of 50 μg / mL kanamycin and 50 μg / mL chloramphenicol) and culture it overnight in a shaker at 37°C and 200 rpm.

[0168] (2) Add all the overnight cultured bacteria into 100 mL of L-M9 liquid culture medium (containing a final concentration of 50 μg / mL kanamycin and 50 μg / mL chloramphenicol) and culture in a shaking incubator at 37°C and 200 rpm for 3 h. At this time, the OD value of the bacterial solution is about 0.6.

[0169] (3) Add IPTG to a final concentration of 0.3 mM and culture at 20°C and 200 rpm for 18 h.

[0170] (4) Then, NaCl or NaBr solution with a final concentration of 300 mM, 0.7% glucose and 1.9 mM tryptophan were added, and the culture was continued at 30°C and 200 rpm in a shaking incubator for 24 h.

[0171] (5) If the fermentation broth of the strain appears purple, it indicates that the corresponding tnaA+ mutant has high selectivity for 6'-halogenated tryptophan.

[0172] Some shake flask screening results are as follows Figure 6 and Figure 7 As shown, Figure 6 This is the result of partial shake flask fermentation rescreening of substrate preference test. The color changes with the halogenation time. After 24 hours of halogenation fermentation, the color is basically stable. Figure 6 The results showed that the control shake flasks of the conventional tryptophanase strain were blue and did not turn purple, indicating that the tryptophanase used the tryptophan substrate to produce indigo and could not use the halogenated tryptophan substrate. However, under the same culture conditions, the other tryptophanase mutant strains showed a significant color change. The shake flasks that turned purple indicated that the corresponding strains used the halogenated tryptophan substrate to produce the Tyler purple pigment, indicating that the corresponding tryptophanase mutants had a preference for the halogenated tryptophan.

[0173] The 6'-halogenated tryptophan-preferring strain obtained in this example was fermented under the same culture conditions. Figure 7 This was a partial shake flask fermentation rescreening. After 48 hours of fermentation, the strain's preference for halogenated tryptophan was initially determined based on the degree of purple color. The main purple component is 6,6'-dibromoindigo or 6,6'-dichloroindigo; the higher the content, the darker the purple. A "+" sign was used to indicate the degree of preference for different mutants. One "+" indicated effective but weak preference, with a light red color; two "+"s indicated moderate effectiveness, with a light purple or purple-red color; three "+"s indicated very effective, with a distinct, single purple color; and "-" indicated ineffectiveness. The statistical results are shown in Table 4.

[0174] Table 4 Statistical results of mutant bias

[0175] Mutant number Test number 50 394 395 Cl substitution preference Br substitution preference 60 1 L S T + - 51 1_3 A P S +++ ++ 47 1_11 S G S ++ + 42 8 V A S +++ +++ 43 3_15 T A S ++ - 45 3_13 C A S ++ - 38 3_4 G T G +++ - 35 3_9 M S G +++ - 29 3 I I G ++ + 26 2_1 L G G ++ ++ 9 2_6 T I C ++ - 12 3_11 L I C +++ ++ 14 5 G I C ++ - 1 1_2 N A A ++ +

[0176] 3. Substrate preference quantitative shake flask fermentation

[0177] (1) Take 10 μL of the 040+ strain stored at -80°C and inoculate it into 4 mL of liquid LB medium (containing a final concentration of 50 μg / mL kanamycin and 50 μg / mL chloramphenicol) and culture it overnight in a shaker at 37°C and 200 rpm.

[0178] (2) Add all the overnight cultured bacteria into 100 mL of L-M9 liquid culture medium (containing a final concentration of 50 μg / mL kanamycin and 50 μg / mL chloramphenicol) and culture in a shaking incubator at 37°C and 200 rpm for 3 h. At this time, the OD value of the bacterial solution is about 0.6.

[0179] (3) Add IPTG to a final concentration of 0.3 mM and culture at 20°C and 2000 rpm for 18 h.

[0180] (4) Then, glucose with a final concentration of 0.7%, 1.0 mM tryptophan chloride or tryptophan bromide, and 1.0 mM tryptophan were added, and the culture was continued at 30°C and 200 rpm in a shaking incubator for 24 h.

[0181] (5) Take 2 mL of bacterial culture and centrifuge at 12,000 rpm for 5 min. Take the supernatant and filter it through a 0.22 μm membrane into a sample bottle. Perform liquid chromatography on the remaining tryptophan and halogenated tryptophan in the sample.

[0182] 1) Prepare standard stock solution:

[0183] Prepare 40mM L-tryptophan, 6-chloro-L-tryptophan and 6-bromo-L-tryptophan stock solutions with ddH2O, dilute the stock solutions with ddH2O, and prepare standards with concentrations of 0mM, 0.25mM, 0.5mM, 1mM, and 2mM, respectively. Detect the retention area at 280nm by HPLC and draw a standard curve. The standard curve for L-tryptophan is as follows: Figure 8 The standard curve of 6-chloro-L-tryptophan is shown in Figure 9 The standard curve of 6-bromo-L-tryptophan is shown in Figure 10 shown.

[0184] 2) Sample testing

[0185] The treated samples were sent for high performance liquid chromatography (HPLC) using a C18 column, a column temperature of 39°C, a flow rate of 1 ml / min, an absorption peak at 280 nm, and mobile phases: A: 0.3% TFA / water B: 0.3% TFA / methanol. The liquid elution gradient is shown in Table 5.

[0186] Table 5 Liquid phase elution gradient

[0187] Time(min) A B 0 95％ 5％ 8 40％ 60％ 10 0％ 100％ 12 95％ 5％ 15 95％ 5％

[0188] According to the liquid phase results, the peak area was calculated and the standard curve formula was used to calculate the content of the substrate L-tryptophan and halogenated tryptophan remaining after the reaction. The results are as follows: Figure 11 and Figure 12 As shown. After 24 hours of reaction, mutants with test numbers 1-3 and 8 still had approximately 0.95 mM tryptophan remaining, while chlorinated tryptophan was essentially consumed, indicating a strong preference for chlorinated tryptophan. Mutant with test number 8 still had approximately 0.96 mM tryptophan remaining after 24 hours of reaction, while brominated tryptophan was essentially consumed, indicating a strong preference for brominated tryptophan. The remaining mutants also showed varying degrees of preference for halogenated tryptophan.

[0189] The amino acid sequence of the Tyler purple powder strain fusion expression: tnaA+-FL-MaFMO, as shown in SEQ ID NO.18. SEQ ID NO.18:

[0190]

[0191] SEQ ID NO. 18 shows the sequence of tnaA+-FL-MaFMO, using one tryptophanase mutant as an example. For different tryptophanase mutants, the sequence can be modified accordingly. In the sequence, single underlining indicates the TnaA amino acid sequence, double underlining indicates the linker sequence, and single wavy underlining indicates the MaFMO amino acid sequence.

[0192] Example 5

[0193] Construction of an engineered strain for the synthesis of 6'-halogenated tryptophan-preferring tyrpuroside and verification by shake flask screening. The specific steps are as follows:

[0194] 1. Construction of an engineered bacterial strain for the one-step fermentation synthesis of 6,6'-dibromoindigoside

[0195] (1) The pSEV331-Ptac-tnaA+-fl-fmo 6'-halotryptophan selective plasmid obtained by screening in Example 5 was used as the backbone for PCR amplification using primers ZT-F1 (SEQ ID NO. 9) and ZF-R1 (SEQ ID NO. 10). The product was recovered by 1% agarose gel electrophoresis and then used as a standby. The gel-recovered product was named Vector. The gel-extraction recovery kit used in this example was the TIANgel Midi Purification Kit DP209. The PCR amplification reaction system and reaction conditions for ZT-F1 and ZF-R1 were the same as those for tnaA-BsaⅠ-F and tnaA-BsaⅠ-R.

[0196] SEQ ID NO.9: 5'-ctgcagcttggactcctgttgatag-3'

[0197] SEQ ID NO.10:

[0198] 5'-CTAGTATTTCTCCTCTTTCTCTAGATTAAACAAAATTATTTGTAGAGGC-3'

[0199] (2) PCR amplification of pSEV331-Ptac-tnaA+-fl-fmo was performed using primers Linker-tanA-F (SEQ ID NO. 3) and ZT-R2 (SEQ ID NO. 11). The product was recovered by 1% agarose gel electrophoresis and then used for future use. The gel-recovered product was named tnaA+-fl-fmo. The gel-cleavage recovery kit used in this example was the TIANgel Midi Purification Kit DP209. The PCR amplification reaction system and reaction conditions for Linker-tanA-F and ZT-R2 were the same as those for tnaA-BsaⅠ-F and tnaA-BsaⅠ-R.

[0200] SEQ ID NO.11: 5'-tctagtatataaacgcagaaaggcc-3'

[0201] (3) The plasmid pW1-Ptac-tnaA-FL-fmo-sarj-UGT (SEQ ID NO. 17) sequence was amplified by PCR using primers Ptac-UGT-F (SEQ ID NO. 7) and Ptac-UGT-R (SEQ ID NO. 8). The product was recovered by 1% agarose gel electrophoresis and then used for future use. The gel-recovered product was named Ptac-UGT. The gel-recovery kit used in this example was the TIANgel Midipurification Kit DP209. The PCR amplification reaction system and reaction conditions for Ptac-UGT-F and Ptac-UGT-R were the same as those for tnaA-BsaⅠ-F and tnaA-BsaⅠ-R. The plasmid pW1-Ptac-tnaA-FL-fmo-sarj-UGT is referenced in patent application CN115851847A.

[0202] SEQ ID NO.7:

[0203] 5'-ggcctttctgcgtttatatactagaGATCaatGGTCTCAGAGAGCAGAGG-3'

[0204] SEQ ID NO.8:

[0205] 5'-ctatcaacaggagtccaagctgcagCCAATCCGGATATAGTTCCTCCTTTCAG-3'

[0206] SEQ ID NO.17:

[0207]

[0208]

[0209] In the sequence shown in SEQ ID NO. 17, the underlined dotted line indicates the Ptac promoter region, the double underline indicates the RiboJ-RBS, the single underline indicates the UGT, and the single wavy underline indicates the terminator region.

[0210] (4) The PCR products Vector, tnaA+-fl-fmo, and Ptac-UGT were assembled using the Gibson assembly method to obtain a ligated plasmid named pSEV331-Ptac-tnaA+-FL-fmo-Ptac-UGT. For the specific assembly reaction steps and parameters, please refer to the Molecular Cloning Laboratory Manual (4th edition).

[0211] (5) The assembled plasmid pSEV331-Ptac-tnaA+-FL-fmo-Ptac-UGT was then transformed into the DH5α(DtnaA)::fre-FL-sttH strain, and the resulting double-plasmid recombinant strain was named 040+-UGT. For specific assembly reaction steps and parameters, refer to the Molecular Cloning Laboratory Manual (4th Edition). The 040+-UGT series strains can use tryptophan and sodium chloride or sodium bromide as precursors to ferment and synthesize the tyril glycoside product in a one-step process.

[0212] Among them, the DH5α(DtnaA)::fre-FL-sttH strain is referenced in patent application CN115851847A.

[0213] The sequence of the strain expressing Tyrpuroside fusion is: Ptac-tnaA+-FL-fmo-Ptac-UGT, as shown in SEQ ID NO. 19. The sequence shown in SEQ ID NO. 19 only lists one tryptophanase mutant. The sequences of the remaining tryptophanase mutants can be changed accordingly according to the specific mutation type.

[0214] SEQ ID NO.19:

[0215]

[0216]

[0217]

[0218] In the sequence, the underlined dotted line indicates the Ptac promoter region, the double underline indicates the RiboJ-RBS, the single underline indicates the tnaA mutant, the bold underlined dotted line indicates the Linker, the double wavy underline indicates fmo, the bold underlined single line indicates the UGT, and the single wavy underline indicates the terminator region.

[0219] 2.040+-UGT strain shake flask fermentation verification

[0220] (1) Take 10 μL of the 040+ strain stored at -80°C and inoculate it into 4 mL of liquid LB medium (containing a final concentration of 50 μg / mL kanamycin and 50 μg / mL chloramphenicol) and culture it overnight in a shaker at 37°C and 200 rpm.

[0221] (2) Add all the overnight cultured bacteria into 100 mL of L-M9 liquid culture medium (containing a final concentration of 50 μg / mL kanamycin and 50 μg / mL chloramphenicol) and culture in a shaking incubator at 37°C and 200 rpm for 3 h. At this time, the OD value of the bacterial solution is about 0.6.

[0222] (3) Add IPTG to a final concentration of 0.3 mM and culture at 20°C and 2000 rpm for 20 h.

[0223] (4) Then, NaCl or NaBr solution with a final concentration of 300 mM, 0.7% glucose, and 1.9 mM tryptophan were added to the 040+-UGT fermentation broth, and the culture was continued at 30°C and 200 rpm in a shaking incubator for 24 h.

[0224] 1 mL of fermentation broth was centrifuged and 0.1 mL of BGL enzyme extract was added and mixed for 1 hour until the solution turned purple. BGL enzyme is referenced in patent application CN115851847A.

[0225] The results indicate that UGT is a glycosylated protein that modifies the expressed pigment. This glycosylated pigment is highly water-soluble, and the solution retains the original color of the fermentation broth without turning purple. BGL enzyme deglycosylates the pigment, converting water-soluble pigment molecules into insoluble pigment granules, resulting in a purple solution. Therefore, the purple color change after the addition of the BGL enzyme extract indicates that the strain is fermenting correctly and is capable of synthesizing the glycosylated pigment.

[0226] Example 6

[0227] One-step synthesis of Tyrian purple powder with a 6'-halogenated tryptophan preference using engineered strain 040+, verified by fermentation in a 10L tank. The specific steps are as follows:

[0228] 1. Inoculation solution: Add 0.40 μg of inoculation solution to 5 mL of LB (50 μg / mL Kana, 50 μg / mL Cm) and culture overnight at 37°C with a shaker at 200 rpm.

[0229] 2. Seed liquid expansion culture: Add all 5 mL of overnight culture into 100 mL LB (50 μg / mL Kana, 50 μg / mL Cm) liquid medium and culture at 37°C in a shaking incubator until the OD reaches 0.8.

[0230] 3.040+10L tank-to-tank induction

[0231] 1) Collect 100 mL of seed solution by centrifugation at 10,000 rpm at room temperature. Resuspend the cells in an equal volume of M9 medium and wash twice, finally resuspending to 100 mL.

[0232] 2) Add the inoculated solution at a ratio of 2% to the fermentation tank containing 5LM9 liquid culture medium.

[0233] 3) Fermentation tank conditions: initial temperature 37° C., rotation speed 200 rpm, pH to 6.9 regulated with 20% ammonia water and 2 M phosphoric acid, aeration rate 0.4 vvm.

[0234] 4) Incubate at 37°C for approximately 3 hours until the OD reaches approximately 0.6. Add IPTG to a final concentration of 0.3 mM for induction. Induction temperature: 20°C, incubate for 18 hours.

[0235] 4.040+ halogenation: Add 300mM NaCl, 1.9mM NaCl or NaBr and 0.7% glucose to the bacterial solution after 040+ protein induction, and culture at 30°C for 24h.

[0236] 5. Collect the bacteria and color powder by centrifugation, then resuspend them in clean water, break the cells by ultrasonic treatment for 50 minutes, centrifuge at 10000 rpm for 10 minutes at room temperature, remove the supernatant, and obtain the Tyler purple powder.

[0237] 6. The collected Tyrian purple powder was dried at 60-70°C and stored at room temperature.

[0238] The toner yield in this example was 698 mg / L in a 10L tank. Some of the fermentation results are shown in the figure below. Figure 13 As shown in the figure, the results of 10L fermentation verification of the engineering bacteria for synthesizing 6'-halogenated tryptophan-biased tyloside are shown. The tryptophanase of this strain is an APS mutant.

[0239] Figure 13The results showed that when tryptophan was added during the fermentation process of the strain, tryptophan would be halogenated and modified by the halogenase. If the fermentation broth was blue, it meant that the tryptophanase expressed by the strain used the tryptophan substrate to synthesize indigo pigment. If the fermentation broth was purple, it meant that the tryptophanase expressed by the strain used the halogenated tryptophan substrate to synthesize 6',6'-dibromoindigo or 6',6'-dichloroindigo pigment, and the resulting color powder was Tyrian purple. Figure 13 As shown, the fermentation liquid is purple, indicating that the strain utilizes halogenated tryptophan and the final product is Tyrian purple powder.

[0240] Example 7

[0241] One-step synthesis of 6'-halogenated tryptophan-biased tyrpuroside using engineered bacteria 040+-UGT, verified by 10L fermentation. The specific steps are as follows:

[0242] 1. Inoculation: Add 040+-UGT inoculum to 5 mL LB (50 μg / mL Kana, 50 μg / mL Cm) and culture overnight at 37°C with a shaker at 200 rpm.

[0243] 2. Seed liquid expansion culture: Add all 5 mL of overnight culture into 100 mL LB (50 μg / mL Kana, 50 μg / mL Cm) liquid medium and culture at 37°C in a shaking incubator until the OD reaches 0.6-1.

[0244] 3.040+-UGT protein induction

[0245] 1) Collect 100 mL of seed solution by centrifugation at 10,000 rpm at room temperature. Resuspend the cells in an equal volume of M9 medium and wash twice, finally resuspending to 100 mL.

[0246] 2) Add the inoculated solution at a ratio of 2% to the fermentation tank containing 5LM9 liquid culture medium.

[0247] 3) Fermentation tank conditions: initial temperature 37°C, rotation speed 500 rpm, associated dissolved oxygen content 30%, pH ~6.9 regulated with 20% ammonia and 2M phosphoric acid, aeration rate 0.5 vvm.

[0248] 4) After 3 h of fermentation, linear feeding of 1×M9 (containing 400 g / L glucose) was started, and 250 mL of linear feeding was added within 15 h.

[0249] 5) Ferment for 24 hours, adjust the temperature to 20°C, and add IPTG to a final concentration of 0.3 mM for induction. Continue induction for 20 hours, then feed the culture at a constant rate of 5 mL / h with 100 mL of 1× M9 (containing 400 g / L glucose).

[0250] 4.040+-UGT halogenation glycosylation

[0251] 1) After protein induction, adjust the temperature to 28°C and start halogenated glycosylation.

[0252] 2) Add 0.4 mM tryptophan, 300 mM NaCl and 0.7% glucose to the bacterial solution after protein induction at one time.

[0253] 3) Then, at an interval of 0.5 h, batch feeding of 0.05 mM tryptophan was started, with a total tryptophan feeding amount of 2.8 mM.

[0254] 5. After 96 hours of fermentation, collect the supernatant glycoside solution by centrifugation.

[0255] 6. Take 200 mL of the glycoside solution obtained by fermentation, add 0.1 mL of BGL enzyme extract and mix well and react for 72 hours. The solution will turn purple.

[0256] The toner was collected and dried at 60-70℃, and the toner yield was counted. The results showed that the toner yield of the glycoside solution obtained in the 10L tank was 1308mg / L. Figure 14 As shown, Figure 14 The results of the one-step synthesis of 6'-halogenated tryptophan-biased tyrpuroside by the engineered strain 040+-UGT in a 10L tank fermentation were verified (the tryptophanase of this strain is a LIC mutant). Figure 14 Figure A is the fermentation diagram of the glycoside solution, Figure B is the BGL reaction process of the glycoside solution, and Figure C is the color powder obtained by the glycoside solution reaction.

[0257] Figure 14 The results showed that when tryptophan was added during the fermentation culture of the strain, the tryptophan would be halogenated and modified by the halogenase. UGT is a glycosylated protein that will glycosylate the expressed pigment. The pigment after glycosylation modification has good water solubility, and the solution will be the original color of the fermentation liquid and will not turn purple. BGL enzyme can deglycosylate. After deglycosylation, the water-soluble pigment molecules will become insoluble pigment particles, and the solution will be purple. Therefore, after the BGL enzyme extract is added, the color turns purple, indicating that the strain ferments correctly and can synthesize the pigment after glycosylation modification. Figure 14 As shown in A, the fermentation liquid is in its original color, indicating that the pigment synthesized by the strain has been modified by glycosylation and has become water-soluble. Figure 14 B is the fermentation broth subjected to BGL enzyme liquid reaction, which is purple, indicating that after deglycosylation, it becomes insoluble pigment particles, and also indicates that the tryptophanase mutant of this strain utilizes halogenated tryptophan. Figure 14 C is the fermentation liquid after BGL enzyme reaction, and the final product obtained by drying is Tyrian purple powder.

[0258] According to the literature doi:10.3390 / 60900736, Tyler purple pigment is soluble in DMSO and has a specific absorption peak at 604nm. This example refers to this method to detect the purity of the final color powder.

[0259] (1) Preparation of standard stock solution:

[0260] Prepare 0.05mM dichloroindigo pigment stock solution with DMSO, dilute the stock solution with DMSO, and prepare standard products with concentrations of 0mM, 0.0025mM, 0.005mM, 0.01mM, 0.0125mM, 0.015mM, 0.02mM, and 0.0225mM, respectively. Take 200μL of the standard product, measure the absorbance at 604nm with a microplate reader, and draw a dichloroindigo standard curve, as shown in the following figure: Figure 15 shown.

[0261] (2) Sample testing

[0262] The collected toner was dried and weighed, then crushed in a mortar. 0.006 g (± 0.0001 g) of toner was weighed and thoroughly mixed with 1 mL of water. 50 μL of the solution was then added to 1 mL of DMSO to fully dissolve the mixture. Finally, 200 μL of the standard solution was taken and the absorbance at 604 nm was measured using a microplate reader. Substituting this into the standard curve formula, the dichloroindigo content in the toner was calculated to be 0.75 mM. This conversion yielded a dichloroindigo content of 0.006 g, resulting in a Tyrian purple powder purity of 82.5%.

[0263] Based on the above experiments, the present application finally screened and verified that 60 three-point mutants with substrate selectivity were obtained by simultaneous mutation of amino acid L at position 50, amino acid V at position 394, and amino acid E at position 395, as shown in Table 2; among them, 14 tryptophanase mutants with better 6'-halogenated tryptophan substrate preference were preferably obtained, as shown in Table 3.

[0264] The above content is a further detailed description of the present application in conjunction with specific implementation methods, and the specific implementation of the present application cannot be considered to be limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A tryptophanase mutant, characterized in that: The tryptophanase is obtained by mutating at least one of the 50th amino acid L, the 394th amino acid V and the 395th amino acid E of the tryptophanase parent.

2. The tryptophanase mutant according to claim 1, characterized in that: The amino acid L at position 50 is mutated to A, V, I, N, S, G, T, C, H, F or M; and / or, the amino acid V at position 394 is mutated to I, S, A, P, T, H, G, N, L or F; And / or, the amino acid E at position 395 is mutated to S, G, C, A, E, T, P or D.

3. The tryptophanase mutant according to claim 2, characterized in that: It is obtained by simultaneously mutating the 50th amino acid L, the 394th amino acid V and the 395th amino acid E of the tryptophanase parent; Preferably, the amino acid L at position 50 is mutated to N, the amino acid V at position 394 is mutated to A, and the amino acid E at position 395 is mutated to A; Alternatively, the amino acid L at position 50 is mutated to H, the amino acid V at position 394 is mutated to A, and the amino acid E at position 395 is mutated to A; Alternatively, the amino acid L at position 50 is mutated to S, the amino acid V at position 394 is mutated to L, and the amino acid E at position 395 is mutated to A; Alternatively, the amino acid L at position 50 is mutated to C, the amino acid V at position 394 is mutated to N, and the amino acid E at position 395 is mutated to A; Alternatively, the amino acid L at position 50 is mutated to A, the amino acid V at position 394 is mutated to S, and the amino acid E at position 395 is mutated to A; Alternatively, amino acid L at position 50 is mutated to V, amino acid V at position 394 is mutated to V, and amino acid E at position 395 is mutated to A; Alternatively, the amino acid L at position 50 is mutated to I, the amino acid V at position 394 is mutated to V, and the amino acid E at position 395 is mutated to A; Alternatively, the amino acid L at position 50 is mutated to V, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to C; Alternatively, the amino acid L at position 50 is mutated to T, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to C; Alternatively, the amino acid L at position 50 is mutated to S, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to C; Alternatively, the amino acid L at position 50 is mutated to N, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to C; Alternatively, the amino acid L at position 50 is mutated to L, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to C; Alternatively, the amino acid L at position 50 is mutated to I, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to C; Alternatively, the amino acid L at position 50 is mutated to G, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to C; Alternatively, the amino acid L at position 50 is mutated to A, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to C; Alternatively, the amino acid L at position 50 is mutated to A, the amino acid V at position 394 is mutated to P, and the amino acid E at position 395 is mutated to C; Alternatively, the amino acid L at position 50 is mutated to N, the amino acid V at position 394 is mutated to T, and the amino acid E at position 395 is mutated to C; Alternatively, amino acid L at position 50 is mutated to V, amino acid V at position 394 is mutated to V, and amino acid E at position 395 is mutated to C; Alternatively, the amino acid L at position 50 is mutated to L, the amino acid V at position 394 is mutated to P, and the amino acid E at position 395 is mutated to D; Alternatively, the amino acid L at position 50 is mutated to S, the amino acid V at position 394 is mutated to A, and the amino acid E at position 395 is mutated to E; Alternatively, the amino acid L at position 50 is mutated to V, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to E; Alternatively, the amino acid L at position 50 is mutated to T, the amino acid V at position 394 is mutated to A, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to I, the amino acid V at position 394 is mutated to A, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to L, the amino acid V at position 394 is mutated to G, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to T, the amino acid V at position 394 is mutated to H, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to A, the amino acid V at position 394 is mutated to H, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to I, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to C, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to L, the amino acid V at position 394 is mutated to P, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to C, the amino acid V at position 394 is mutated to P, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to V, the amino acid V at position 394 is mutated to S, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to N, the amino acid V at position 394 is mutated to S, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to M, the amino acid V at position 394 is mutated to S, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to L, the amino acid V at position 394 is mutated to S, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to V, the amino acid V at position 394 is mutated to T, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to G, the amino acid V at position 394 is mutated to T, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to I, the amino acid V at position 394 is mutated to V, and the amino acid E at position 395 is mutated to G; Alternatively, amino acid L at position 50 is mutated to G, amino acid V at position 394 is mutated to V, and amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to V, the amino acid V at position 394 is mutated to G, and the amino acid E at position 395 is mutated to P; Alternatively, the amino acid L at position 50 is mutated to V, the amino acid V at position 394 is mutated to A, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to T, the amino acid V at position 394 is mutated to A, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to F, the amino acid V at position 394 is mutated to A, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to C, the amino acid V at position 394 is mutated to A, and the amino acid E at position 395 is mutated to S; Alternatively, amino acid L at position 50 is mutated to C, amino acid V at position 394 is mutated to F, and amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to S, the amino acid V at position 394 is mutated to G, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to I, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to N, the amino acid V at position 394 is mutated to P, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to L, the amino acid V at position 394 is mutated to P, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to A, the amino acid V at position 394 is mutated to P, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to V, the amino acid V at position 394 is mutated to S, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to N, the amino acid V at position 394 is mutated to S, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to L, the amino acid V at position 394 is mutated to S, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to H, the amino acid V at position 394 is mutated to S, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to G, the amino acid V at position 394 is mutated to T, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to T, the amino acid V at position 394 is mutated to V, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to G, the amino acid V at position 394 is mutated to V, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to S, the amino acid V at position 394 is mutated to A, and the amino acid E at position 395 is mutated to T; Alternatively, the amino acid L at position 50 is mutated to L, the amino acid V at position 394 is mutated to S, and the amino acid E at position 395 is mutated to T; Preferably, the amino acid L at position 50 is mutated to N, the amino acid V at position 394 is mutated to A, and the amino acid E at position 395 is mutated to A; Alternatively, the amino acid L at position 50 is mutated to T, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to C; Alternatively, the amino acid L at position 50 is mutated to L, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to C; Alternatively, the amino acid L at position 50 is mutated to G, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to C; Alternatively, the amino acid L at position 50 is mutated to L, the amino acid V at position 394 is mutated to G, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to I, the amino acid V at position 394 is mutated to I, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to M, the amino acid V at position 394 is mutated to S, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to G, the amino acid V at position 394 is mutated to T, and the amino acid E at position 395 is mutated to G; Alternatively, the amino acid L at position 50 is mutated to V, the amino acid V at position 394 is mutated to A, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to T, the amino acid V at position 394 is mutated to A, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to C, the amino acid V at position 394 is mutated to A, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to S, the amino acid V at position 394 is mutated to G, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to A, the amino acid V at position 394 is mutated to P, and the amino acid E at position 395 is mutated to S; Alternatively, the amino acid L at position 50 is mutated to L, the amino acid V at position 394 is mutated to S, and the amino acid E at position 395 is mutated to T.

4. The tryptophanase mutant according to any one of claims 1 to 3, characterized in that: The tryptophanase parent is the sequence shown in SEQ ID NO.

12.

5. A nucleic acid encoding the tryptophanase mutant according to any one of claims 1 to 4.

6. A recombinant plasmid containing the nucleic acid according to claim 5.

7. A microorganism transformed with the recombinant plasmid according to claim 6.

8. The microorganism according to claim 7, characterized in that: The tryptophanase mutant was co-expressed by fusion with the flavin monooxygenase MaFMO via a linker; Preferably, the tryptophanase mutant is co-expressed by fusion with the flavin monooxygenase MaFMO-UGT via a linker.

9. Use of the tryptophanase mutant according to any one of claims 1 to 4, the nucleic acid according to claim 5, the recombinant plasmid according to claim 6, or the microorganism according to claim 7 or 8 in the preparation of Tyrian purple pigment.

10. Tyrian purple pigment prepared using the tryptophanase mutant according to any one of claims 1 to 4, the nucleic acid according to claim 5, the recombinant plasmid according to claim 6, or the microorganism according to claim 7 or 8.

11. A method for screening tryptophanase mutants with a bias towards 6'-halogenated tryptophan, characterized in that: The following steps are included: In the protein model construction step, the molecular 3D structures of tryptophanase, coenzyme pyridoxal phosphate and tryptophan were docked using software to obtain the protein model of TnaA+PLP+Trp; The mutation site analysis step includes using software to modify the TnaA+PLP+Trp model and analyze the mutation site; The mutation library construction step involves performing saturation random mutagenesis on the mutation sites obtained through analysis to obtain a tryptophanase mutation library; In the tryptophanase mutant screening step, the tryptophanase mutant library is screened at least once using a tryptophan substrate to obtain a tryptophanase mutant with a 6'-halogenated tryptophan preference.

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